200948395 六、發明說明： 【發明所屬之技術領域】 本申請案係關於適用於藉由離子電滲向生物體皮内投與 蛋白質之方法的組合物，其中組合物包含帶負電之蛋白 質-脂質體複合物。 本申δ月案根據35 U.S.C· § 119(e)規定主張2008年8月14 曰申4之美國臨時專利申請案第61/088,939號之權益；其 中此臨時申請案以引用的方式全部併入本文中。 ❹ 【先前技術】 表皮層中富含抗原呈現細胞（例如，蘭氏細胞 (Langerhans cell))，該細胞在免疫系統中產生重要作用。 皮内疫苗可比其他投與途徑更有效地向目標細胞（諸如， 抗原呈現細胞）傳遞抗原。 皮内注射一般已用作皮内投與抗原之方法。近年來，已 研發出藉由物理力（諸如針、壓迫或電流之力（例如，顯微 針、喷霧注射器或電穿孔））在皮膚上形成小孔來傳遞抗原 之方法。然而，迄今研發出的皮内傳遞抗原之投與方法一 般由於（例如）需要必須將皮膚角質層穿刺至一定程度（例 如，使用喷霧注射器時）或需要向患者皮膚施加高電壓（例 如，使用電穿孔時）而與患者疼痛有關。此外，相較於藉 由皮内注射投與抗原而言’難以藉由經由纖維針、噴霧注 射器或電穿孔投與抗原誘發足夠免疫反應。因此，迄今研 發之皮内投與疫苗之方法在安全性及有效性方面均存在問 題。 138820.doc 200948395 另一方面，藉由向已將預定部分施加於生物體皮膚上之 離子型藥物施加電動力來經生物體之皮膚或黏膜（本文總 稱為「皮膚」）引入（亦即，滲透）離子型藥物之方法係稱為 離子電滲（例如，參見JP 63_35266 Α)β近年來，已揭示離 子電滲為向生物體投與藥物之非侵襲性且安全之方法。 在離子電滲中，具有正電荷之離子型化合物一般藉由電 動力之陽極側推斥而推進（亦即，輸送）穿過及/或進入生物 鱧皮膚。另一方面，具有負電荷之離子型化合物一般藉由 電動力之陰極侧推斥而推進（亦即，輸送）穿過及/或進入生 物體皮膚。 舉例而言，Marro，D 等人，讲 2001年12月，第18卷，第12期，第17〇117〇8頁報導在藥 物藉由離子電滲自陰極側投與之狀況下，藥物之投與效率 由於自皮膚内側至外側的離子滲流（水流）而降低；且在藥 物自陽極側投與之狀況下，藥物藉由電力及離子滲流有效 地皮内傳遞。 此外，在PCT/JP20〇7/〇71368中已表明藥物可藉由自離 子電滲裝置之陽極側投與囊封該藥物之陽離子脂質體有效 地皮内傳遞。然自本申請案之申請者的實驗可見，已 闡明甚至自離子電滲之陽極側投與囊封大抗原蛋白質之陽 離子月曰質體亦難以有效地皮内傳遞抗原蛋白質。 【發明内容】 本文揭示藉由離子電滲向生物體有效地皮内投與具有大 分子量之蛋白質的方法，其中蛋白質在生物體中有效地誘 138820.doc 200948395 發免疫反應。 申請者已發現當藉由離子電滲向生物體投與由具有抗原 性之蛋白質及陽離子脂質體形成之帶負電複合物時，可顯 著促進蛋白質之皮内傳遞且可有效誘發免疫反應《本申請 案之實施例係基於該等發現。 因此’本申請案之一實施例提供離子電滲用之組合物， 其能有效地皮内傳遞蛋白質且有效誘發免疫反應。 該離子電滲用之組合物包含帶負電之蛋白質-脂質體複200948395 VI. Description of the Invention: [Technical Field] The present application relates to a composition suitable for a method of intradermally administering a protein to an organism by iontophoresis, wherein the composition comprises a negatively charged protein-liposome Complex. This application is based on 35 USC § 119(e) and claims the US Provisional Patent Application No. 61/088,939 of August 14, 2008; this provisional application is incorporated by reference. In this article. ❹ [Prior Art] The epidermal layer is rich in antigen-presenting cells (for example, Langerhans cells), which play an important role in the immune system. Intradermal vaccines can deliver antigens to target cells, such as antigen presenting cells, more efficiently than other routes of administration. Intradermal injection has generally been used as a method of intradermal administration of antigen. In recent years, methods have been developed to deliver antigens by forming small holes in the skin by physical forces such as needles, compression or current (e.g., microneedles, spray syringes or electroporation). However, the methods of administration of intradermal delivery antigens developed to date generally arise, for example, from the need to puncture the stratum corneum of the skin to a certain extent (eg, when using a spray syringe) or the need to apply a high voltage to the patient's skin (eg, using When electroporated) is associated with patient pain. Furthermore, it is difficult to induce a sufficient immune response by administering an antigen via a fiber needle, a spray injector or electroporation as compared to administration of an antigen by intradermal injection. Therefore, the methods of intradermal administration of vaccines developed to date have problems in terms of safety and efficacy. 138820.doc 200948395 On the other hand, by the application of electric power to an ionic drug that has applied a predetermined portion to the skin of a living body, it is introduced through the skin or mucous membrane of the living body (collectively referred to herein as "skin") (ie, infiltration). The method of the ionic drug is called iontophoresis (for example, see JP 63_35266 Α). In recent years, iontophoresis has been disclosed as a non-invasive and safe method of administering a drug to an organism. In iontophoresis, an ionic compound having a positive charge generally propels (i.e., transports) through and/or into the skin of the living body by repulsion of the anode side of the electromotive force. On the other hand, an ionic compound having a negative charge is generally advanced (i.e., transported) through and/or into the skin of a living body by repulsion of the cathode side of the electromotive force. For example, Marro, D et al., December 2001, Vol. 18, No. 12, pp. 17〇117〇8 report on the drug administered by iontophoresis from the cathode side. The administration efficiency is lowered by ion permeation (water flow) from the inner side to the outer side of the skin; and in the case where the drug is administered from the anode side, the drug is efficiently delivered intradermally by electric power and ion permeation. Furthermore, it has been shown in PCT/JP 20 〇 7/〇 71368 that the drug can be effectively delivered intradermally by administering the cationic liposome encapsulating the drug from the anode side of the iontophoresis device. However, it can be seen from the experiment of the applicant of the present application that it has been difficult to efficiently deliver the antigenic protein intradermally from the anode side of the iontophoresis to the cation cation of the large antigenic protein. SUMMARY OF THE INVENTION Disclosed herein is a method for efficiently intradermally administering a protein having a large molecular weight to an organism by iontophoresis, wherein the protein effectively induces an immune response in the organism 138820.doc 200948395. Applicants have found that when a negatively charged complex formed by an antigenic protein and a cationic liposome is administered to an organism by iontophoresis, the intradermal delivery of the protein can be significantly promoted and the immune response can be effectively induced. The examples are based on such findings. Thus, an embodiment of the present application provides a composition for iontophoresis which is capable of efficiently delivering proteins intradermally and effectively eliciting an immune response. The composition for iontophoresis comprises a negatively charged protein-liposome complex
&物’其中蛋白質-脂質體複合物係由帶負電之蛋白質及 陰離子脂質體形成。 該離子電滲用之組合物甚至可允許具有相對較大分子量 之蛋白質（諸如，抗原蛋白質）藉由離子電滲有效地皮内傳 遞且因此可有效地誘發免疫反應。此外，該離子電滲用之 組合物在與佐劑一起使用時可能能夠顯著誘發生物體之免 疫反應。 【實施方式】 在圖式中；^同參考數字識別類似元件或操作。圖式中 之冗件的尺寸及相對位置未必按比㈣製。舉例而言，各 種兀件之形狀及角度未按比例繪製，且此等元件中之某些 經：意地放大及定位以改良圖式易讀性。另外，所繪製： =特:：狀不意欲傳達關於特定元件之實際形狀的任 僅為了圖式中之認識簡易性 在以下描述中，M.甘 運释 闡述某些特定細節以提供對各種所福示 實施例之充分轉n ” '為邳關枝術者將認識到實施 138820.doc 200948395 =可無需此等特定細節中之—或多者，或以其他方法、成 刀材料等進行實踐。在其他狀況下，未展示或詳細描述 、皮内傳遞裝置及離子電滲有關之熟知機構以避免實施例 之不必要的模糊描述。 除非本文另有規定，否則說明書及隨後之申請專利範圍 全文中，詞語「包含」及其變體將以開放、包含性之意義 來解釋，亦即解釋為「包括（但不限於）」。 本說明書全文中對「一實施例」之提及意謂、结合該實施 J插述之特疋特徵、結構或特性包括於至少一個實施例 中。因此，在本說明書全文各處之短語「在一實施例中」 的出現未必均指相同實施例。此外，可在一或多個實施例 中以任何合適方式組合特定特徵、結構或特性。 如本說明書及隨附申請專利範圍中所用，除非文中明確 =指示為相反’否則單數形式「一」及「該」包括複數個 指不物。亦應注意，除非文中明確指示為相反，否則術語 「或」一般在其包括「及/或」之意義上使用。 本文所提供之揭示内容之標題及摘要僅為方便起見且不 解釋實施例之範疇及含義。 如本文及申請專利範圍中所用，術語「陽離子脂質體」 意謂脂質體在所選阳值(諸如，生理pH值)下具有淨正電 荷。 如本文及申請專利範圍中所用，術語「陽離子脂質」意 謂脂質在所選pH值（諸如，生理1>1^值）下具有淨正電荷。〜 如本文及中請專利範圍中所用，術語「脂肪酸」可為飽 138820.doc -6 · 200948395 和或不飽和’及直鏈、支鏈或環狀脂肪酸。 離子電滲用之組合物 ::於藉由離子電滲向生物體皮内傳遞蛋白質之組合物 ::铷,負電之蛋白質-脂質體複合物’其中蛋白質-脂質體 ;口糸由帶負電之蛋白質及陰離子脂質體形成。當藉由 夕向生物體皮膚投與帶負電之複合物時’儘管複合 物具有比帶負電蛋白皙 會暗辟女 蛋白質大的分子量，但複合物藉由跨越皮 9 ❹ 土效地皮内滲透。適用於藉由離子電渗向生物體皮 蛋白質之經合物亦可包含除帶負電蛋白質脂質體 複0物之外的成分。 蛋白質-脂質體複合物 根據至7自實施例，蛋白質-脂質體複合物係由帶負 電蛋白質及陰離子脂質體形成，且整個複合物之淨電荷為 負蛋白質-月曰質體複合物可藉由將蛋白質及脂質體置於 能產生電荷相互作用之系統内且使蛋白質與脂質體聚集形 成蛋白質-脂質體複合物而形成…般而言，蛋白質-脂質 體複合物係藉由制靜電相互作闕為主要㈣力使蛋白 質與脂質體結合而形成。 帶負電蛋白質-脂質體複合物具有較佳_50至-5 mV且更 佳-40至-10 mV之ζ電位。 帶負電蛋白質與陽離子脂質體之正電荷的負/正（-/+)電 荷比可鑒於複合物之形成料㈣當敎，且較佳為Η = 10:1 ’且更佳為3:1至8:1。電荷比係基於以下原理來計 异：脂質體總電荷的一半係用作脂質體電荷，因為脂質體 I38820.doc 200948395 係由脂質雙層膜形成且雙層膜内之 肤正電何未參與靜電相互 作用。舉例而言’在脂質體由具有 電何之单價陽離子脂 質形成的狀況下，-/+比可藉由以下等式來計算· (-/+電荷比）=[(蛋白質之量（monw 里Vmoi))x(蛋白質中 數）]:[陽離子脂質之量（m〇1))/2] 订〜 [等式1] 蛋白質之量及陽離子脂質之量可易於根據負載量及其類 似量來測定。 蛋白質-脂質體複合物之平约船；古> J <十岣拉子直徑不受特別限制， 只要蛋白質可藉由離子電滲虔内玺 电β反内傳遞，且較佳為1〇〇至 10，_ nm，且更佳為i，000至1〇,〇〇〇⑽。給出動填光散 射、靜態光散射、以電鏡觀測及以原子力顯微鏡觀測作為 測疋平均粒子直控之方法。即使蛋白質.脂質體複合物具 有如上所述之粒度’複合物亦可藉由離子電滲向生物體皮 内投與且在向生物體皮内投與大蛋白質(諸如，抗原蛋白 質）方面具有優勢。 帶負電之蛋白質 適用於蛋白質，㈣複合物之帶負電蛋白質與陽離子 脂=體組合在能產生電荷相互作用之线中形成複合物。 帶負電之蛋白質不受特別限制，只要蛋白f帶負電且可 與腊質體形成複合物且較佳在3至狀邱值下，更佳在4至 9之pH值下帶負電。 帶負電蛋白質可為由各具有負電荷之相同胺基酸形成的 蛋白質，且可為由兩種或兩種以上各具有負電荷之不同胺 138820.doc 200948395 基酸形成的蛋白暂 ,,.._ ^ ^ *白質。此外，蛋白質可包括各具有正電荷之 及各具有負電荷之胺基酸，只要蛋白質具有淨負電 荷。 ' 帶負電蛋白質令負電荷總數較佳為1至100,更佳為5至 負電荷總數可由熟習此項技術者藉由蛋白質中各具 有負電何之胺基酸數目及各具有正電荷之胺基駿數目之間 的差異來測定。 ❹ :負電蛋白質之分子量不受特別限制，只要帶負電蛋白 質可藉由蛋白質·脂f體複合物經皮内傳遞，較佳為3，_ 心至1〇〇，_ kDa，更佳5，_ kDa至50，_ kDae甚至可 傳遞具有上述分子量之蛋白質。因此，蛋白質-脂質體複 合物可有利地用於傳遞具有相對較大分子量之蛋白質，諸 如抗原蛋白質。 " 帶負電蛋白質較佳可用作抗原。帶負電蛋白質之實例包 括（但不特定限於）印白蛋白（下文稱為「〇VA」）、人類免 疫缺陷病毒(HIV)之GAG蛋白、人類流感病毒之血球凝集 素及神經胺酸酶，及B型肝炎病毒之HBs抗原蛋白。 陽離子鹿質趙 適用於蛋白質-脂質體複合物之陽離子脂f體具有淨正 電荷且可與帶負電蛋白質組合形成帶負電蛋白質_脂質體 複合物。 陽離子脂質體之平均粒子直徑不受特別限制，只要蛋白 質可皮内傳遞’且較佳為50至nm，且更佳為職 500請。敎平均粒子直徑之方法與測定複合物之粒子直 138820.doc 200948395 徑的方法相同β 具有淨正電荷之脂質體包含至少陽離子脂質作為構成成 分°陽離子脂質較佳為具有1至10價正電荷之C12_C20脂 質’更佳為具有1至3價正電荷之C14-C20脂質，且更佳為 具有1價正電荷之C14-C18脂質《陽離子脂質之特定實例包 括（但不限於）1,2-二油醯氧基_3·(三甲基敍基）丙烧 (DOTAP)、二（十八烷基）二甲基氣化銨（d〇dac)、N-(2,3-二油烯氧基）丙基-N，N，N-三甲基銨（DOTMA)、二（十二烷 基）漠化銨（DDAB)、1,2-二肉豆蔻醯氧基丙基―丨义二甲基 經基乙基銨（DMRIE)及2,3-二油醯氧基_n_[2(精胺甲醯胺） 乙基]-N，N-二甲基-1-丙基三氟乙酸銨（D〇SPA)。較佳為 1，2-二油酿氧基-3-(三甲基銨）丙烷（d〇TAP)、二（十八烷基） 二甲基氣化銨（DODAC)、N-(2,3-二油醯氧基）丙基_n,N，N-二甲基銨（DOTMA)及2,3-二油醯氧基_N-[2(精胺甲醯胺）乙 基]-N，N-二甲基-1-丙基三氟乙酸銨（D〇SPA)。更佳為12-二油醯氧基-3-(三曱基鍵）丙院（DOTAP)。 此外，脂質體較佳進一步包含固醇、磷脂或其組合。 固醇可根據陽離子脂質體之穩定性、蛋白質-脂質體複 合物之傳遞效率及其類似因素來適當選擇，且較佳為膽固 醇（Choi)、膽固醇基脂肪酸、二氫膽固醇基脂肪酸或膽固 醇醚’且更佳為膽固酵（Choi)、C12-C31膽固醇基脂肪 酸、C12-C31二氫膽固醇基脂肪酸、聚氧化乙烯膽固醇醚 及聚氧化乙烯二氫膽固醇醚，且又佳為膽固醇（Ch〇1)。 磷脂可根據蛋白質-脂質體複合物之傳遞效率來適當選 138820.doc -10- 200948395 擇且較佳為C12-C20磷脂且更佳為C14-C18磷脂。磷脂之 特定實例包括（但不限於）磷脂醯膽鹼、卵磷脂（EPC)、二棕 櫚醯基磷脂醯膽鹼（DPPC)及麟脂醯乙醇胺。較佳為二硬脂 醯基L-α-磷脂醯膽鹼（DSPC)、卵磷脂（EPC)、二棕櫚醯基 鱗脂醯膽驗（DPPC)或其組合。更佳為二硬脂醯基L_a_麟脂 醯膽鹼（DSPC)。 0&' wherein the protein-liposome complex is formed from a negatively charged protein and an anionic liposome. The composition for iontophoresis can even allow a protein having a relatively large molecular weight (such as an antigenic protein) to be efficiently intradermally delivered by iontophoresis and thus can effectively induce an immune response. Furthermore, the composition for iontophoresis may be capable of significantly inducing an immunological reaction of an organism when used together with an adjuvant. [Embodiment] In the drawings, the same reference numerals are used to identify similar elements or operations. The size and relative position of the redundant parts in the drawings are not necessarily in accordance with (4). For example, the shapes and angles of various components are not drawn to scale, and some of these components are intended to be enlarged and positioned to improve the legibility of the drawings. In addition, the drawing: = special:: is not intended to convey the actual shape of a particular component. It is merely an understanding of the simplicity of the drawing. In the following description, M. Gan explained some specific details to provide The full rotation of the exemplified embodiment n "" will be recognized by the practitioners of the implementation of 138820.doc 200948395 = may not be required in any of these specific details - or more, or by other methods, knife materials, etc. In other instances, well-known mechanisms that are not shown or described in detail, intradermal delivery devices, and iontophoresis are used to avoid unnecessary ambiguous description of the embodiments. Unless otherwise stated herein, the specification and subsequent claims are incorporated by reference in their entirety. The word "including" and its variants will be interpreted in terms of openness and inclusiveness, which is interpreted as "including (but not limited to)". Reference throughout the specification to "an embodiment" means that the features, structures, or characteristics described in connection with the embodiment J are included in at least one embodiment. Thus, appearances of the phrases "in an embodiment" Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. As used in the specification and the appended claims, the singular forms "a" It should also be noted that the term "or" is generally used in its sense including "and/or" unless the context clearly indicates otherwise. The title and abstract of the disclosure provided herein are for convenience only and do not explain the scope and meaning of the embodiments. As used herein and in the scope of the patent application, the term "cationic liposome" means that the liposome has a net positive charge at a selected positive value (such as physiological pH). As used herein and in the scope of the patent application, the term "cationic lipid" means that the lipid has a net positive charge at a selected pH, such as a physiological 1>1 value. ~ As used herein and in the scope of the patent, the term "fatty acid" may be 138820.doc -6 · 200948395 and or unsaturated ' and linear, branched or cyclic fatty acids. Composition for iontophoresis: a composition for transferring protein to an organism by iontophoresis: 铷, a negatively charged protein-liposome complex, wherein the protein-liposome; the sputum is negatively charged Protein and anionic liposomes are formed. When a negatively charged complex is administered to the skin of the living body by the eve, although the complex has a larger molecular weight than the negatively charged peptone, the complex penetrates through the skin. The composition suitable for ionophoresis to the biological skin protein may also contain components other than the negatively charged protein liposome complex. Protein-Liposome Complex According to 7 from the embodiment, the protein-liposome complex is formed by a negatively charged protein and an anionic liposome, and the net charge of the entire complex is a negative protein - the ruthenium complex can be obtained by Proteins and liposomes are formed in a system that generates charge interactions and aggregates proteins and liposomes to form protein-liposome complexes. In general, protein-liposome complexes are made by electrostatic interaction. It is formed by combining the protein with the liposome for the main (four) force. The negatively charged protein-liposome complex has a zeta potential of preferably _50 to -5 mV and more preferably -40 to -10 mV. The negative/positive (-/+) charge ratio of the negative charge of the negatively charged protein to the cationic liposome may be 敎, and preferably Η = 10:1 ' and more preferably 3:1, in view of the formation of the composite (4) 8:1. The charge ratio is based on the following principle: half of the total charge of the liposome is used as the liposome charge, because the liposome I38820.doc 200948395 is formed by the lipid bilayer membrane and the skin in the bilayer membrane is not involved in the static electricity. interaction. For example, in the case where the liposome is formed by a monovalent cationic lipid having electricity, the -/+ ratio can be calculated by the following equation: (-/+ charge ratio) = [(the amount of protein (monw) Vmoi))x (protein number)]: [cationic lipid amount (m〇1))/2] 订~ [Equation 1] The amount of protein and the amount of cationic lipid can be easily determined according to the amount of load and the like. Determination. The protein-liposome complex is not limited to a specific diameter as long as the protein can be transferred by iontophoresis, and preferably 1 〇. 〇 to 10, _ nm, and more preferably i, 000 to 1 〇, 〇〇〇 (10). Dynamic fill light scattering, static light scattering, electron microscopy and atomic force microscopy are used as methods for measuring the average particle direct control. Even if the protein. liposome complex has the particle size 'complex as described above, it can be administered intradermally to the organism by iontophoresis and has an advantage in administering large proteins (such as antigenic proteins) into the skin of the organism. . Negatively charged proteins are suitable for proteins, and (4) complexes with negatively charged proteins and cationic lipids = body combinations form complexes in lines that generate charge interactions. The negatively charged protein is not particularly limited as long as the protein f is negatively charged and forms a complex with the waxy body and is preferably negatively charged at a pH of 4 to 9 and preferably at a pH of 4 to 9. The negatively charged protein may be a protein formed by the same amino acid having a negative charge, and may be a protein formed by two or more different amines having a negative charge of 138820.doc 200948395 acid, ... _ ^ ^ * White matter. Further, the protein may include an amino acid each having a positive charge and a negative charge, as long as the protein has a net negative charge. The negatively charged protein preferably has a total number of negative charges of from 1 to 100, more preferably from 5 to a total number of negative charges, which can be obtained by those skilled in the art by the number of amino acids each having a negative charge in the protein and each having a positively charged amine group. The difference between the number of horses is measured. ❹ : The molecular weight of the negatively charged protein is not particularly limited as long as the negatively charged protein can be intradermally delivered by the protein·lipid complex, preferably 3, _ heart to 1 〇〇, _ kDa, more preferably 5, _ kDa to 50, _ kDae can even deliver a protein having the above molecular weight. Therefore, the protein-liposome complex can be advantageously used to deliver a protein having a relatively large molecular weight, such as an antigenic protein. " Negatively charged proteins are preferably used as antigens. Examples of negatively charged proteins include, but are not particularly limited to, albumin (hereinafter referred to as "〇VA"), human immunodeficiency virus (HIV) GAG protein, human influenza virus hemagglutinin and neuraminidase, and HBs antigenic protein of hepatitis B virus. Cationic Deer Quality Zhao The cationic liposome suitable for the protein-liposome complex has a net positive charge and can be combined with a negatively charged protein to form a negatively charged protein-liposome complex. The average particle diameter of the cationic liposome is not particularly limited as long as the protein can be intradermally delivered 'and preferably 50 to nm, and more preferably 500. The method of measuring the average particle diameter of the crucible is the same as the method of measuring the particle diameter of the composite 138820.doc 200948395. The liposome having a net positive charge contains at least a cationic lipid as a constituent component. The cationic lipid preferably has a positive charge of 1 to 10 valence. The C12_C20 lipid is more preferably a C14-C20 lipid having a positive charge of 1 to 3, and more preferably a C14-C18 lipid having a monovalent positive charge. Specific examples of the cationic lipid include, but are not limited to, 1,2-di Oil 醯oxy_3·(trimethylxyl)propane (DOTAP), di(octadecyl)dimethylammonium hydride (d〇dac), N-(2,3-dioleyloxy) Propyl-N,N,N-trimethylammonium (DOTMA), di(dodecyl)ammonium (DDAB), 1,2-dimyristyloxypropyl-pyroline Base ethylethylammonium (DMRIE) and 2,3-dioleyloxy_n_[2 (spermine methotrexate) ethyl]-N,N-dimethyl-1-propyltrifluoroacetate (D〇SPA). Preferred is 1,2-dioleyloxy-3-(trimethylammonium)propane (d〇TAP), di(octadecyl)dimethylammonium hydride (DODAC), N-(2, 3-dioleyloxy)propyl-n,N,N-dimethylammonium (DOTMA) and 2,3-dioleyloxy_N-[2(sperminecarbamamine)ethyl]- N,N-Dimethyl-1-propylammonium trifluoroacetate (D〇SPA). More preferably, it is a 12-dioleyloxy-3-(trimethylene bond) propyl hospital (DOTAP). Further, the liposome preferably further comprises a sterol, a phospholipid or a combination thereof. The sterol can be appropriately selected depending on the stability of the cationic liposome, the delivery efficiency of the protein-liposome complex, and the like, and is preferably cholesterol (Choi), cholesterol-based fatty acid, dihydrocholesteryl fatty acid or cholesterol ether. More preferably, it is Choi, C12-C31 cholesterol-based fatty acid, C12-C31 dihydrocholesteryl fatty acid, polyoxyethylene cholesterol ether, and polyoxyethylene dihydrocholesterol ether, and is preferably cholesterol (Ch〇1). ). The phospholipid may be appropriately selected according to the transfer efficiency of the protein-liposome complex, and is preferably a C12-C20 phospholipid and more preferably a C14-C18 phospholipid. Specific examples of phospholipids include, but are not limited to, phospholipid choline, lecithin (EPC), dipalmitoside phospholipid choline (DPPC), and linoleum ethanolamine. Preferred are distearyl decyl L-α-phospholipid choline (DSPC), lecithin (EPC), dipalmitosyl sphincter (DPPC) or a combination thereof. More preferably, it is a distearyl group L_a_liner choline (DSPC). 0
此外’在脂質體除作為構成成分的陽離子脂質之外亦包 含磷脂、固醇或其組合之狀況中，陽離子脂質與磷脂及固 醇之總和的莫耳比可根據皮内傳遞效率或其類似因素來適 當測定，且較佳為1:9至9:1，且更佳為上8至心2。此外， 在脂質體包含磷脂及固醇兩者的狀況下，磷脂與固醇之莫 耳比較佳為2:8至8:2 ’且更佳為3:7至7:3。此外，根據一 只施例冑離子脂質、碟脂與固醇之間的莫耳比為約 2:5:3 。 在本文所揭tf之方法中，蛋白質脂質體複合物可用作 離子電滲用之組合物。细人队„八π 物組合物除蛋白質-脂質體複合物之 外亦可包含一或多種成公， /、要額外成分不妨礙藉由離子 電渗投與蛋白質-脂質體複合物。 額外成分不受特別限制，σ J ，、要額外成分不妨礙藉由離子 電滲投與蛋白質-脂質體滿人机 體複β物。額外成分之實例包 不限於）水及醫藥學上可接 接又之載劑’諸如緩衝劑（諸如 HEPES)、防腐劑、增溶劍 W如 及著色劑。 ”“劑、穩定劑、抗氧化劑 只要不妨礙藉由離子電渗投與蛋白質-脂質體複合物， 138820.doc 200948395 包含蛋白質-脂質體複合物之組合物可視需要形成為適當 調配物。舉例而言，可以乾燥形式形成包含蛋白質-脂質 體複合物之組合物。舉例而言，鑒於藉由離子電滲有效投 與複合物，組合物較佳與水或HEPES缓衝劑組合形成溶 液。在此狀況下，離子電滲用之組合物的pH值（例如）為7 至8。此外’組合物之離子強度為（例如”至“爪“。 蛋白質-脂質體複合物在組合物中之含量可視需要進行 適當測定。 製造方法 蛋白質-脂質體複合物可易於藉由使蛋白質與陽離子脂 質體混合且使混合物聚集來形成。 θ 舉例而言，製備用於形成蛋白質_脂質體複合物之脂質 體。可藉由熟習相關技術者已知的方法或藉由本 之方法來製備脂質體。 揭 舉例而言，將陽離子脂質、磷脂及固醇以所要比率在諸 如水之液體介質巾混合，藉此獲得混合溶液。接著，在減 壓下移除介質’且因此獲得脂質膜。隨後，向脂質膜中添 加緩衝劑，例如HEPES緩衝劑（1〇至5〇 mM)。使所獲得之 混合溶液在室溫下靜置㈣min以使混合溶液水合，=後 進行超音波處理。超音波處理之條件例如（但不限於^ W’室溫’及約i min。此外，將混合溶液經膜過濾器、擠 壓機或其類似裝置處理以調節所要粒子直徑，藉此獲得脂 質體》 隨後 製備含有帶負電蛋白質 之第一水溶液及含有脂質 138820.doc 200948395 體之第二水溶液。 帶負電蛋白質在第一水溶液中之濃度及脂質體在第二水 /合液中之濃度係根據各蛋白質及脂質體對溶劑的溶解度、 帶負電蛋白質-脂質體複合物之形成效率及其類似因素來 適當測定 pH值、離子強度及第一與第二水溶液之溫度可根據蛋白 質與知質體之充電狀態，及最終複合物之形成效率來適當 調節。Further, in the case where the liposome contains a phospholipid, a sterol or a combination thereof in addition to the cationic lipid as a constituent component, the molar ratio of the cationic lipid to the total of the phospholipid and the sterol may be based on the intradermal delivery efficiency or the like. It is suitably determined, and is preferably from 1:9 to 9:1, and more preferably from 8 to 2. Further, in the case where the liposome contains both a phospholipid and a sterol, the phospholipid and the sterol molar are preferably 2:8 to 8:2' and more preferably 3:7 to 7:3. Further, according to one embodiment, the molar ratio between the ionic lipid, the dish grease and the sterol is about 2:5:3. In the method of tf disclosed herein, the protein liposome complex can be used as a composition for iontophoresis. The squad „ eight π composition may contain one or more of the granules in addition to the protein-liposome complex, and the additional component does not interfere with the protein-liposome complex by iontophoresis. Without particular limitation, σ J , additional components are not hindered by iontophoresis and protein-liposome filling of human body complex β. Examples of additional components are not limited to water and pharmaceutically acceptable Carriers such as buffers (such as HEPES), preservatives, solubilizing swords such as and coloring agents. "" Agents, stabilizers, antioxidants, as long as they do not interfere with the application of protein-liposome complexes by iontophoresis, 138820.doc 200948395 A composition comprising a protein-liposome complex can be formed into a suitable formulation as desired. For example, a composition comprising a protein-liposome complex can be formed in a dry form. For example, in view of ionizing electricity The osmosis is effective to administer the complex, and the composition is preferably combined with water or HEPES buffer to form a solution. In this case, the pH of the composition for iontophoresis is, for example, from 7 to 8. Further The ionic strength of the substance is (for example, to "claw". The content of the protein-liposome complex in the composition can be appropriately determined as needed. Manufacturing method Protein-liposome complex can be easily mixed by mixing the protein with the cationic liposome And the mixture is aggregated to form. θ For example, a liposome for forming a protein-liposome complex is prepared. The liposome can be prepared by a method known to a person skilled in the art or by the method of the present invention. That is, the cationic lipid, the phospholipid, and the sterol are mixed at a desired ratio in a liquid medium towel such as water, thereby obtaining a mixed solution. Then, the medium is removed under reduced pressure and thus a lipid film is obtained. Subsequently, into the lipid film A buffer such as HEPES buffer (1 〇 to 5 〇 mM) is added. The obtained mixed solution is allowed to stand at room temperature for (four) min to hydrate the mixed solution, and then subjected to ultrasonic treatment. The conditions of the ultrasonic treatment are, for example ( However, it is not limited to ^ W 'room temperature' and about i min. In addition, the mixed solution is treated by a membrane filter, an extruder or the like to adjust the desired particles. Path, thereby obtaining a liposome. Subsequently, a first aqueous solution containing a negatively charged protein and a second aqueous solution containing a lipid 138820.doc 200948395 are prepared. The concentration of the negatively charged protein in the first aqueous solution and the liposome in the second water/combination The concentration in the liquid is determined according to the solubility of each protein and liposome to the solvent, the formation efficiency of the negatively charged protein-liposome complex, and the like, and the pH, the ionic strength, and the temperature of the first and second aqueous solutions can be appropriately determined. The state of charge of the protein and the plastid, and the formation efficiency of the final complex are appropriately adjusted.
第一及第二水溶液中所用之溶劑較佳為水及缓衝劑，且 更佳為水、HEPES緩衝劑及其類似物。 將第水溶液及第二水溶液混合在一起獲得混合液體。 混。方法不受特別限制。可將第二水溶液添加至第一水溶 液中’且或者’可將第—水溶液添加至第二水溶液中。此 外’可將第-性水溶液及第二水溶液同時添加至容器中以 便混合。所獲得之第一水溶液與第二水溶液之混合液體可 經適當攪拌。 笫一 及第二水溶液之混合比可經適當測定以便根據帶負 電蛋白質與陽離子脂f體在混合液體中之_/+電荷比形成具 有過量負電荷之蛋白質-脂質體複合物。舉例而言，可設The solvent used in the first and second aqueous solutions is preferably water and a buffer, and more preferably water, HEPES buffer and the like. The aqueous solution and the second aqueous solution are mixed together to obtain a mixed liquid. Mixed. The method is not particularly limited. The second aqueous solution may be added to the first aqueous solution 'and or' the first aqueous solution may be added to the second aqueous solution. Further, the first aqueous solution and the second aqueous solution may be simultaneously added to the container to be mixed. The mixed liquid of the obtained first aqueous solution and the second aqueous solution can be appropriately stirred. The mixing ratio of the first aqueous solution and the second aqueous solution can be suitably determined to form a protein-liposome complex having an excessive negative charge based on the _/+ charge ratio of the negatively charged protein to the cationic lipid f body in the mixed liquid. For example, it can be set
定混合比以使得第—氽：交、沐+ > A '液中之負電荷總數超過第二水溶 液中之正電荷總數。此外 m 外兩>谷液中之電荷數可易於根據 用於製備兩溶液之蛋白皙弋 質或陽離子脂質之莫耳比及淨電荷 數來設定。 此外，混合液體之pH值 離子強度及溫度可根據複合物 138820.doc -13- 200948395 之形成效率來適當測$。混合液體之pH值及離子強度 由改變上文之第-水溶液及第二水溶液的組成(例如，濃 度、量、pH值及離子強度）及混合比來調節。特定言之: 混合液體之pH值為（例如）3至10。此外，混合液體I離子 強度為（例如）5至20 mM » 混合液體之溫度為（例如）i6它至4〇°c。 混合液體可原樣靜置且使其中產生蛋白質_脂質體複合 物，其中混合液體可用作離子電滲用之組合物。在其中= 成蛋白質-脂質體複合物之前，混合液體較佳經培育。/ ^ 培月混合液體之條件可為例如（但不限於）16〇c至; 及 15 至 60 min。 經培育之混合液體可經離心分離以獲得聚集物。離心分 離條件可為例如（但不限於）3〇〇〇至1〇〇〇〇Xg ; 2它至6它； 及 5 至 1〇 min。 將例如（但不限於）水或緩衝劑之水性溶劑添加至所獲得 之聚集物中，藉此可獲得離子電滲用之含有帶負電蛋白 質-脂質體複合物之組合物。離子電滲用之組合物可由熟 0 習相關技術者使用緩衝劑或其類似物調節以具有有利pH值 及適於帶負電複合物之離子強度。在此狀況下，根據帶負 電複&物之ζ電位或其類似因素，較佳地調節pH值及離子 強度。 水性、溶劑之溫度可由熟習相關技術者根據蛋白質脂質 體複合物之形成效率來適當測定，且（例如）為16°C至 40〇C。 138820.doc •14· 200948395 應用 藉由離子電滲將蛋白質-脂質體複合物及其組合物應用 至生物體且較佳用作在生物體中誘發免疫反應之疫苗。此 外，蛋白質-脂質鳢複合物及其組合物可用於離子電渗以 有效地皮内傳遞對生物體具有抗原性之蛋白質，其中有效 誘發生物體之免疫反應。因此，蛋白質_脂質體複合物及 其組合物較佳係用作皮内疫苗製劑。 電極組合（assemb丨y)及離子電滲裝置The mixing ratio is set such that the total amount of negative charges in the first - 氽: 交, 沐 + > A ' liquid exceeds the total amount of positive charges in the second aqueous solution. Further, the number of charges in the m outer two > trough liquid can be easily set depending on the molar ratio and the net charge number of the protein tantalum or cationic lipid used to prepare the two solutions. Further, the pH of the mixed liquid ionic strength and temperature can be appropriately measured by the formation efficiency of the composite 138820.doc -13 - 200948395. The pH and ionic strength of the mixed liquid are adjusted by changing the composition of the first aqueous solution and the second aqueous solution (e.g., concentration, amount, pH, and ionic strength) and the mixing ratio. Specifically: The pH of the mixed liquid is, for example, 3 to 10. Further, the mixed liquid I ion intensity is, for example, 5 to 20 mM » The temperature of the mixed liquid is, for example, i6 to 4 ° C. The mixed liquid can be left as it is and a protein-liposome complex is produced therein, wherein the mixed liquid can be used as a composition for iontophoresis. The mixed liquid is preferably cultivated before the = protein-liposome complex. / ^ The conditions for mixing the liquid may be, for example, but not limited to, 16 〇c to; and 15 to 60 minutes. The incubated mixed liquid can be centrifuged to obtain aggregates. The centrifugation separation conditions may be, for example, but not limited to, 3 Torr to 1 Torr Xg; 2 it to 6 Å; and 5 to 1 〇 min. An aqueous solvent such as, but not limited to, water or a buffer is added to the obtained aggregate, whereby a composition containing a negatively charged protein-liposome complex for iontophoresis can be obtained. The composition for iontophoresis can be adjusted by a skilled person using a buffer or the like to have an advantageous pH and an ionic strength suitable for the negatively charged complex. In this case, the pH and the ionic strength are preferably adjusted depending on the zeta potential of the negatively charged & The temperature of the aqueous solution and the solvent can be suitably determined by a person skilled in the art based on the formation efficiency of the protein liposome complex, and is, for example, 16 ° C to 40 ° C. 138820.doc •14· 200948395 Application The protein-liposome complex and its composition are applied to an organism by iontophoresis and are preferably used as a vaccine for inducing an immune response in an organism. Further, the protein-lipid ruthenium complex and the composition thereof can be used for iontophoresis to efficiently deliver an antigenic protein to an organism, which effectively induces an immune response of the organism. Therefore, the protein-liposome complex and its composition are preferably used as an intradermal vaccine preparation. Electrode assembly (assemb丨y) and iontophoresis device
此外，可藉由使用固持離子電滲用之組合物的電極組合 及裝備有該電極組合之離子電滲裝置來有利地進行蛋白 質-脂質體複合物向生物體之投與。 根據-實施例’提供一電極組合，其包括電極、用於固 持組合物之蛋白質-脂質體複合物固持部分，肖固持部分 係置於電極之皮膚侧，纟中蛋白質_脂質體複合物可藉由 離子電渗釋放至生物體古患。山从 王物镀皮廣。此外，只要不妨礙藉由離子 電滲投與蛋白質-脂質錄滿人私> 舶買體複合物，可另外在電極與蛋白質_ 脂質體複合物固持部分之間提供電解質溶液固持部分。 /此外’蛋白質·脂質體複合物帶負電，所以較佳向電力 糸統之陰極側施加倉雪、4 貝電流。因此，在該實施例之電極組合Further, the administration of the protein-liposome complex to the living body can be advantageously carried out by using an electrode combination for holding the composition for iontophoresis and an iontophoresis device equipped with the electrode combination. According to the embodiment, an electrode combination is provided, which comprises an electrode, a protein-liposome complex holding portion for holding the composition, and a Xiao retaining portion is placed on the skin side of the electrode, and the protein-liposome complex in the sputum can be borrowed Released by iontophoresis to ancient organisms. The mountain is widely plated from the king. Further, an electrolyte solution holding portion may be additionally provided between the electrode and the protein-liposome complex holding portion as long as it does not interfere with the protein-lipid recording by the iontophoresis. Further, the 'protein-liposome complex is negatively charged. Therefore, it is preferable to apply a snow and a 4-bar current to the cathode side of the power system. Therefore, the electrode combination in this embodiment
中，電極係指陰核β A ^ 此外’較佳使用由諸如碳或鉑之導電 材料形成之電極作盎蕾士 作為電極。彼等材料亦可用於如下文所述 之反電極中》 蛋白質-脂質體複合物 成之單元（電極室）形成， 固持部分可由丙烯或其類似物製 其係由組合物浸潰、固持組合物 138820.doc -15 - 200948395 ,以組合物填充。或者，蛋白質脂質體複合物固持部分 可由非編織物、脫脂棉或薄膜體形成，其由組合物浸潰且 固持組合物。作為薄膜體之構成元件，具有有利浸潰-固 持特徵及有利離子傳遞特性之材料較佳。材料之實例包括 丙烯酸樹脂之水凝膠體(丙烯酸水凝膠膜)及基於嵌段聚胺 基甲酸s旨之凝膠膜。上述單元及薄膜體亦可用於構成電解 質溶液固持部分。 此外，離子電滲裝置之構成可經適當改變，只要離子電 滲裝置包括電極組合且能夠投與蛋白質_脂質體複合物。 離子電渗裝置較佳包括至少—個電源單元，與㈣單元之 陰極連接之電極組合，及與電源單元之陽極連接之電極組 合。 與陽極連接之電極組合之結構可以適當改變，只要該電 極組合可用作固持蛋白質-脂質體複合物之電極組合之反 電極。舉例而言，作為反電極之電極組合可具有與陽極連 接之電極組合相同的構成，除蛋白質_脂質體複合物固持 部分改變為電解質溶液固持部分之外。根據—個實施例， 作為反電極之電極組合包括至少一個陽極。此外，根據一 個較佳實施例，作為反電極之電極組合進一步包括電解質 溶液固持部分，其係提供於陽極之皮膚侧且固持電解質溶 液。 皮内投舆蛋白質之方法/在生物體中誘發免疫反應之方法 一種包含蛋白質-脂質體複合物之組合物可藉由上述離 子電滲裝置或其類似物向生物體皮内投與該組合物而顯著 138820.doc -16- 200948395 誘發該生物體對該蛋白質的免疫反應。因此，根據另一實 施例’提供一種誘發生物體對蛋白質之免疫反應的方法， 該方法包含藉由離子電滲向生物體皮内投與有效劑量之由 蛋白質及陽離子脂質體形成之帶負電蛋白質-脂質體複合 物。 在離子電滲中’可根據蛋白質-脂質體複合物之投與效 率適當決定通電條件。電流值較佳為0.1至0.45 mA/ cm2， 更佳為 〇·1 至 0·2 mA/cm2。 此外’在生物體中誘發免疫反應之方法中，較佳另外向 生物體投與有效劑量之佐劑以改良生物體對蛋白質的免疫 反應。佐劑可在離子電滲之前或之後向生物體連續投與， 或可與蛋白質-脂質體複合物同時投與。投與佐劑之方法 不受特別限制，只要該方法不妨礙佐劑之作用。舉例而 言，有一種向生物體之施加離子電滲的皮膚部分施加佐劑 之方法。 佐劑為熟習相關技術者已知。較佳佐劑為皮内佐劑。更 佳佐劑為單磷脂A、募核酸序列（所謂CpG)、脂多醣（所謂 LPS)、胞壁醯二肽（MDp)、牛分枝桿菌細胞壁（bcg_cws) 及其類似物。更佳為單磷脂A。 在生物體令有效誘發免疫反應之蛋白質_脂質體複合物 或佐劑的劑量係由熟習此項技術者根據生物體之疾病種 類、物種、性別、年齡、體重及狀態、投藥計劃及其類似 因素適當測定。 「生物體」較佳為哺乳動物，更佳為人類、牛、豬、 138820.doc 17 200948395 馬、綿羊、狗或貓，且又佳為人類。 下文中，蛋白質-脂質體複合物之實施例、包含蛋白質-脂質體複合物之組合物及其藉由離子電滲之皮内投與方法 係以實例方式詳細描述。應瞭解本發明不受此等實例限 制。 實例1 1. OVA之螢光標記 將於硼酸鹽緩衝溶液中含有10 mg OVA(SIGMA Aldrich) 之溶液與2.8111^溶解於10〇4101；^中之>^8-若丹明 (Rhodamine)混合，隨後在室溫下反應1 h。使所獲得之混 合液體經受使用Sephadex-G100之凝膠過濾以分離游離 NHS-若丹明，藉此獲得經若丹明標記之OVA。 2. OVA-脂質體複合物之製備 以 2/5/3(DOTAP/DSPC/Chol)之比率將 DOTAP、DSPC 及 Choi混合入有機溶劑（諸如，CHC13)中，藉此獲得溶液（總 脂質重量為1.6 mg)。在減壓下移除有機溶劑，且重複有機 溶劑之添加及有機溶劑在減壓下的移除以產生脂質薄膜。 接著，向脂質薄膜中添加0.5 ml 10 mM HEPES緩衝劑以便 脂質總濃度為5 mM，隨後在室溫下水合歷時10 min。接 著，將所得混合物在浴槽型超音波儀中超音波處理，且獲 得脂質體（下文稱為「DSPC脂質體」）溶液。 將150 μΐ 100 mg/ml藉由使用經若丹明標記之OVA所製 備之OVA水溶液添加至500 μΐ DSPC脂質體溶液中。將所 獲得之混合液體在室溫下培育30 min且在5,000xg及4°C下 138820.doc -18· 200948395 離心分離5 min。將所獲得之離心塊懸浮於150 μΐ 10 mM HEPES緩衝劑中，藉此獲得OVA-脂質體複合物溶液。 參考實例1 : OVA囊封之脂質體的製備 以 4/4/2(DOTAP/EPC/Chol)之比率將 DOTAP、卵磷脂（下 文稱為「EPC」）及Choi在有機溶劑（諸如CHC13)中混合， 藉此獲得溶液（總脂質重量為8.3 mg)。在減壓下蒸餾除去 有機溶劑，且接著重複有機溶劑之添加及有機溶劑在減壓 © 下的移除以產生脂質薄膜。接著，向脂質薄膜中添加含有 5 mg/ml Alexa 448標記之 OVA(Invitrogen)之 1 ml 乙酸鹽缓 衝劑（pH 4.5)以便脂質總濃度為12.5 mM，隨後在室溫下水 合歷時10 min。隨後，在所得混合物於浴槽型超音波儀中 超音波處理後，將所得混合物冷凍且解凍（6χ)以產生溶 液。在使用1，000-nm膜擠壓溶液後，接著將所得物在 80,000>^及4°(：下離心分離30分鐘以移除游離0¥入。將所 獲得之離心塊懸浮於300 μΐ乙酸鹽緩衝劑中，藉此獲得 參 OVA囊封之脂質體溶液。 測試實例1 .將OVA-脂質體複合物或OVA囊封之脂質體的溶液填充 於專用小杯中，藉此以Zetasizer®量測粒子直徑及ζ電位。 結果顯示於表1中。OVA-脂質體複合物帶負電，而OVA 囊封之脂質體帶正電。在此狀況下，OVA-脂質體複合物 中OVA之負電荷與脂質體之正電荷的-/+電荷比經計算為 7:1 [(OVA莫耳數X負電荷數）：（DOTAP莫耳數xl/2)]。 138820.doc -19- 200948395 表1:粒子直徑及Γ電位的量測結果 平均粒子直徑(nm) ζ電位(mV) OVA-脂質體複合物 5,285±4,240 -20.06±5.81 OVA囊封之脂質體 545±351 53.75士 6.01 *表1中之數值表示平均值土標準差 測試實例2 : 離子電滲 將OVA-脂質體複合物或OVA囊封之脂質體藉由離子電 滲根據以下程序向SD大鼠（10週大，雄性）投與。 離子電滲裝置 OVA-脂質體複合物帶負電，藉此自陰極側投與。用於 OVA-脂質體複合物投藥之離子電滲裝置顯示於圖1中。 在圖1中，將離子電滲裝置1置於皮膚5上且其係由電源 單元2、固持複合物之電極組合3及作為其反電極之電極組 合4形成。彼等電極組合與導電路徑6及7連接。電極組合3 係由陰極3 1、在陰極3 1之皮膚側提供的蛋白質-脂質體複 合物固持部分32形成。另一方面，電極組合4係由陽極 41、在陽極41之皮膚側提供且固持200 μΐ電解質溶液之電 解質固持部分42形成。此外，蛋白質-脂質體複合物固持 部分32及電解質溶液固持部分42各自係由浸潰有siRNA-聚 陽離子複合物溶液或電解質溶液的非編織物或脫脂棉形 成。 此外，在投與OVA囊封之脂質體之狀況下，OVA囊封之 脂質體帶正電，藉此自陽極側投與。 138820.doc -20· 200948395 OVA-脂質體複合物之投與 離子電滲裝置之抗原固持部分係以200 μΐ實例1之OVA-脂質體複合物溶液來填充。接著，將離子電滲裝置安裝於 SD大鼠（10週大，雄性）之背部皮膚上，該大鼠已在 Nembutal®麻醉下以剪毛機剃毛。接著，在0.45 mA(0.15 mA/cm2)之恆定電流下進行離子電滲歷時60 min。 OVA囊封之脂質體的投與 離子電滲裝置之抗原固持部分（在陽極側）係以200 μΐ參 ® 考實例1之OVA-囊封之脂質體溶液來填充。接著，將離子 電滲裝置安裝於SD大鼠（10週大，雄性）之背部皮膚上，該 大鼠已在Nembutal®麻醉下以剪毛機剃毛。接著，在0.45 mA(0.15 mA/cm2)之恆定電流下進行離子電滲歷時60 min 〇 皮膚組織切片之製備及CLSM觀測 在通電後3 h切除皮膚，且將所得物冷凍且植入最佳切 割溫度（OCT)化合物中。使用冷凍切片機自冷凍皮膚樣本 參 製備量測為20 μηι之切片且以反向共焦雷射掃描顯微鏡 (LSM510 Carl Zeiss)來觀測其。 .因此，如圖2A所示，在OVA囊封之脂質體自陽極側投與 之狀況下，除了在角質層之外，在表皮層或真皮層中幾乎 未觀測到螢光。另一方面，在OVA-脂質體複合物自陰極 侧投與之狀況下，如圖2B中所示，在表皮層及真皮層中觀 測到螢光。 測試實例3 138820.doc -21 · 200948395 OVA囊封之脂質體及OVA_脂質艎複合物的製備 使用無螢光標記之OVA且藉由與實例1及參考實例1相同 之程序來製備OVA囊封之脂質體及OVA-脂質體複合物。 離子電滲之經皮免疫性 藉由離子電滲以與測試實例2相同之程序向SD大鼠之背 部皮膚投與所製備之OVA囊封之脂質體或OVA-脂質體複合 物。第一次投與1週後，藉由離子電滲再次以相同程序1投 與OVA囊封之脂質體或OVA-脂質體複合物。第二次投與1 週後對血液取樣且藉由離心分離收集血清。 藉由ELISA法評估抗OVA抗體之量 藉由ELISA法根據以下程序評估所獲得血清中抗OVA抗 體之產量。 將溶解於碳酸鹽緩衝劑（pH 9·5)中之0.01 mg/ml OVA以 50 μΐ/孔添加至96孔微量滴定盤（nunc immunoplate maxisorp corting)中，隨後在4°C下培育隔夜。隨後，藉由 使用ImmunowashTM(BIO-RADTM)，將培養盤以洗務緩衝劑 (-)(1L PBS+2.1 g NaCl)洗滌3次，且以150 μΐ/孔添加阻斷 劑（1%酪蛋白+1%明膠+0.5%BSA碳酸鹽緩衝溶液），隨後 在37°C下培育1 h。此後，以與上文所述相同之方式將培 養盤以洗滌緩衝劑（+)(洗滌緩衝劑（-)+0.05%氚核-X)洗滌3 次。接著，將各自逐步稀釋10倍至20,000倍之血清樣本添 加至孔中。在37°C下培育1 h後，以與上文所述相同之方 式將培養盤以洗滌緩衝劑（+)洗滌3次。向各孔中添加50 μΐ 經HRP標記之二次抗體。在37°C下培育1 h後，以與上文所 138820.doc -22- 200948395 述相同之方式將培養盤以洗滌緩衝劑（+)洗滌3次，且向各 孔中添加50 μΐ TMB以引起顯色。在室溫下培育10 min 後，向各孔中添加50 μΐ 1 N硫酸以停止顯色反應。在450 nm下以微定量盤讀取器（BIO-RAD™)量測吸光度。 藉由自所量測之吸光度值減去未處理大鼠之血清中的吸 光度值，且藉由將等於或大於偵測限值之血清樣本稀釋度 (吸光度為0.08)用作指數來評估抗OVA抗體之產量。 結果顯示於圖3中。在OVA囊封之脂質體自陽極側投與 β 之狀況下，血清中之抗OVA抗體在所有稀釋度下均完全未 偵測到（ND :未偵測到）。另一方面，在OVA-脂質體複合 物自陰極側投與之狀況下，甚至在約300倍稀釋度（平均值 土標準差）下亦偵測到抗OVA抗體，藉此確認誘發抗體產 生。 測試實例4 1. OVA-脂質體複合物之製備 使用無螢光標記之OVA且藉由與實例1中相同之程序製 ❹ 備OVA-脂質體複合物。 2. 藉由離子電滲以及單磷脂A(MPL)的經皮免疫性 .在經剪毛機剃毛之大鼠背部皮膚上塗覆1 00 μΐ市售MPL 佐劑後，以與測試實例2相同之程序藉由離子電滲投與 OVA-脂質體複合物。進行相同測試，但未塗覆MPL作為對 照。第二次離子電滲1週後對血液取樣且藉由離心分離收 集血清。 藉由ELISA法評價抗OVA抗體之產量 138820.doc -23- 200948395 藉由ELISA法根據與測試實例3相同之程序量測及評估 抗體產量。 結果顯示於圖4中。在即將進行離子電滲之前向抗原投 與部分塗覆MPL之狀況下，抗體產量（平均值）約為未使用 MPL狀況的4倍。此外，在塗覆MpL之狀況下即使血清 經稀釋1，000倍或U00倍以上，亦顯著偵測到抗〇va抗 體。 所說明實施例之上述描述，包括摘要中所述者，不欲為 詳盡的或將實施例限制為所揭示之精確形式。儘管本文為 說明之目的描述特定實施例及實例，但如彼等熟習相關技 術者瞭解，在不悖離本揭示案精神及範疇之狀況下可進行 多種等效修正。本文提供之多個實施例之教示可應用於其 他傳遞方法，而不必為上文一般揭示之例示性離子電滲皮 内傳遞方法* ' 可將上文所述之多個實施例組合以提供其他實施例。就 與本文之特定教示及定義不一致而言，本說明書中所提及 及/或申請案資料表中所列之所有美國專利、美國專利申 請公開案、美國專射請案、外國專利、相專利申請案 及非專利公開案以引用的方式完全併入本文中。若需要 則實施例之態樣可採用多個專利、申請案及公開案之系 統、流程及概念來修正以提供其他實施例。 可根據上文之詳細描述對實施例進行此等及其他改變。 一般而"T，在以下申請專利範圍中，所用術語不應解釋為 將申請專利範圍限制於說明t及中請專利範圍中所揭示之 138820.doc -24- 200948395 特定實施例，但應解釋為包括所有可能實施例以及該等申 請專利範圍授權之完整範疇的等效物。因此，申請專利範 圍不受揭示内容限制。 【圖式簡單說明】 圖1為展示用於在活體内測試中根據一所說明之實施例 投與OVA-脂質體複合物之離子電滲裝置的示意圖； 圖2A為OVA-脂質體複合物投藥後大鼠冷凍皮膚樣本的 反向共焦雷射掃描顯微鏡相片； ® 圖2B為OVA-囊封之脂質體投藥後大鼠冷凍皮膚樣本的 反向共焦雷射掃描顯微鏡相片； 圖3展示與已投與OVA-脂質體複合物或OVA囊封之脂質 體之大鼠血清中抗OVA抗體之產量有關的ELISA結果；及 圖4展示與一起投與單磷脂A(MPL)及OVA-脂質體複合物 之大鼠血清中抗OVA抗體之產量有關的ELISA結果。 【主要元件符號說明】 1 離子電滲裝置 2 電源單元 3 固持複合物之電極組合 4 作為固持複合物之電極組合的反電極之電 極組合 5 皮膚 6 導電路徑 7 導電路徑 31 陰極 138820.doc -25- 200948395 32 41 42 蛋白質-脂質體複合物固持部分 陽極 電解質溶液固持部分 138820.doc -26-In the meantime, the electrode refers to the nucleus β A ^ In addition, it is preferable to use an electrode formed of a conductive material such as carbon or platinum as an electrode. The materials may also be used in a counter-electrode as described below. The protein-liposome complex is formed into a unit (electrode chamber), and the holding portion may be made of propylene or the like, which is impregnated and held by the composition. 138820.doc -15 - 200948395, filled with the composition. Alternatively, the protein liposome complex retaining moiety may be formed from a non-woven fabric, a cotton wool or a film body which is impregnated with the composition and holds the composition. As the constituent elements of the film body, a material having favorable impregnation-holding characteristics and favorable ion transport characteristics is preferable. Examples of the material include an acrylic resin hydrogel (acrylic hydrogel film) and a gel film based on a block polyaminocarboxylic acid. The above unit and the film body can also be used to constitute a holding portion of the electrolyte solution. Further, the constitution of the iontophoresis device can be appropriately changed as long as the iontophoresis device includes an electrode combination and is capable of administering a protein-liposome complex. Preferably, the iontophoresis device comprises at least one power supply unit in combination with an electrode connected to the cathode of the (four) unit and an electrode connected to the anode of the power supply unit. The structure of the electrode combination with the anode may be appropriately changed as long as the electrode combination can be used as a counter electrode for holding the electrode combination of the protein-liposome complex. For example, the electrode combination as the counter electrode may have the same composition as the electrode combination connected to the anode, except that the protein-liposome complex holding portion is changed to the electrolyte solution holding portion. According to one embodiment, the electrode combination as the counter electrode comprises at least one anode. Further, according to a preferred embodiment, the electrode combination as the counter electrode further includes an electrolyte solution holding portion which is provided on the skin side of the anode and holds the electrolyte solution. Method for intradermal administration of a protein / method for inducing an immune reaction in an organism A composition comprising a protein-liposome complex can be administered intradermally to the organism by the above-described iontophoresis device or the like Significantly 138820.doc -16-200948395 induced the organism's immune response to the protein. Thus, according to another embodiment, a method for inducing an immune response to a protein by an organism comprising intradermally administering an effective dose of a negatively charged protein formed by a protein and a cationic liposome to the organism by iontophoresis is provided. - liposome complexes. In the iontophoresis, the energization conditions can be appropriately determined depending on the administration efficiency of the protein-liposome complex. The current value is preferably from 0.1 to 0.45 mA/cm2, more preferably from 〇·1 to 0·2 mA/cm2. Further, in the method of inducing an immune response in an organism, it is preferred to additionally administer an effective dose of an adjuvant to the organism to improve the immune response of the organism to the protein. The adjuvant may be administered to the organism continuously before or after iontophoresis, or may be administered simultaneously with the protein-liposome complex. The method of administering the adjuvant is not particularly limited as long as the method does not hinder the action of the adjuvant. By way of example, there is a method of applying an adjuvant to a portion of the skin to which the iontophoresis is applied. Adjuvants are known to those skilled in the art. A preferred adjuvant is an intradermal adjuvant. More preferred adjuvants are monophospholipid A, nucleic acid sequence (so-called CpG), lipopolysaccharide (so-called LPS), cell wall dipeptide (MDp), Mycobacterium bovis cell wall (bcg_cws) and the like. More preferably, it is a single phospholipid A. The dosage of the protein-liposome complex or adjuvant which is effective in inducing an immune response in an organism is determined by the skilled person according to the disease type, species, sex, age, weight and state of the organism, the administration plan, and the like. Appropriate determination. The "organism" is preferably a mammal, more preferably a human, a cow, a pig, 138820.doc 17 200948395 horse, sheep, dog or cat, and preferably a human. Hereinafter, examples of protein-liposome complexes, compositions comprising protein-liposome complexes, and intradermal administration methods by iontophoresis are described in detail by way of examples. It is to be understood that the invention is not limited by the examples. Example 1 1. Fluorescent labeling of OVA A solution containing 10 mg of OVA (SIGMA Aldrich) in a borate buffer solution was mixed with 2.8111^ dissolved in 10〇4101;^>^8-Rhodamine Then, it was reacted at room temperature for 1 h. The obtained mixed liquid was subjected to gel filtration using Sephadex-G100 to separate free NHS-rhodamine, thereby obtaining rhodamine-labeled OVA. 2. Preparation of OVA-Liposome Complex DOTAP, DSPC and Choi are mixed into an organic solvent (such as CHC13) at a ratio of 2/5/3 (DOTAP/DSPC/Chol), thereby obtaining a solution (total lipid weight) Is 1.6 mg). The organic solvent was removed under reduced pressure, and the addition of the organic solvent was repeated and the organic solvent was removed under reduced pressure to give a lipid film. Next, 0.5 ml of 10 mM HEPES buffer was added to the lipid film so that the total lipid concentration was 5 mM, followed by hydration at room temperature for 10 min. Next, the resulting mixture was ultrasonicated in a bath type ultrasonic apparatus, and a liposome (hereinafter referred to as "DSPC liposome") solution was obtained. 150 μΐ 100 mg/ml was added to the 500 μΐ DSPC liposome solution by using an aqueous solution of OVA prepared with rhodamine-labeled OVA. The obtained mixed liquid was incubated at room temperature for 30 min and centrifuged at 5,000 x g and 4 ° C for 138820.doc -18·200948395 for 5 min. The obtained centrifugation block was suspended in 150 μM of 10 mM HEPES buffer, whereby an OVA-liposome complex solution was obtained. Reference Example 1: Preparation of OVA-encapsulated liposomes DOTAP, lecithin (hereinafter referred to as "EPC") and Choi in an organic solvent (such as CHC13) in a ratio of 4/4/2 (DOTAP/EPC/Chol) Mixing, thereby obtaining a solution (total lipid weight of 8.3 mg). The organic solvent was distilled off under reduced pressure, and then the addition of the organic solvent was repeated and the organic solvent was removed under reduced pressure to give a lipid film. Next, 1 ml of an acetate buffer (pH 4.5) containing 5 mg/ml Alexa 448-labeled OVA (Invitrogen) was added to the lipid film so that the total lipid concentration was 12.5 mM, followed by hydration at room temperature for 10 min. Subsequently, after the resulting mixture was ultrasonically treated in a bath type ultrasonic apparatus, the resulting mixture was frozen and thawed (6 Torr) to give a solution. After the solution was extruded using a 1,000-nm film, the resultant was then centrifuged at 80,000 > and 4° for 30 minutes to remove free 0. The obtained centrifuge block was suspended at 300 μΐ. In the acetate buffer, a liposome solution encapsulated with OVA was obtained. Test Example 1. A solution of OVA-liposome complex or OVA encapsulated liposome was filled in a dedicated cuvette, whereby Zetasizer® was used. The particle diameter and zeta potential were measured. The results are shown in Table 1. The OVA-liposome complex was negatively charged, while the OVA encapsulated liposomes were positively charged. Under this condition, the OVA-liposome complex was negative for OVA. The -/+ charge ratio of the charge to the positive charge of the liposome was calculated to be 7:1 [(OVA mole number X negative charge number: (DOTAP mole number xl/2)]. 138820.doc -19- 200948395 1: Measurement of particle diameter and zeta potential Average particle diameter (nm) ζ potential (mV) OVA-liposome complex 5, 285 ± 4, 240 -20.06 ± 5.81 OVA encapsulated liposome 545 ± 351 53.75 ± 6.01 * Table 1 The values in the mean soil standard deviation test example 2: iontophoresis by OVA-liposome complex or OVA encapsulated liposome Iontophoresis was administered to SD rats (10 weeks old, male) according to the following procedure: Iontophoresis device OVA-liposome complex was negatively charged, thereby being administered from the cathode side. For OVA-liposome complex administration The iontophoresis device is shown in Fig. 1. In Fig. 1, the iontophoresis device 1 is placed on the skin 5 and is composed of a power supply unit 2, an electrode assembly 3 for holding the composite, and an electrode combination as a counter electrode thereof. 4. The electrode combinations are connected to the conductive paths 6 and 7. The electrode combination 3 is formed by the cathode 31 and the protein-liposome complex holding portion 32 provided on the skin side of the cathode 31. On the other hand, the electrode combination 4 is formed by an anode 41, an electrolyte holding portion 42 which is provided on the skin side of the anode 41 and holds a 200 μΐ electrolyte solution. Further, the protein-liposome complex holding portion 32 and the electrolyte solution holding portion 42 are each impregnated with siRNA. - a non-woven or absorbent cotton solution of a polycationic complex solution or an electrolyte solution. Further, in the case of administration of OVA-encapsulated liposomes, the OVA-encapsulated liposomes are positively charged, thereby being cast from the anode side. 138820.doc -20· 200948395 OVA-Liposome Complex The antigen-holding portion of the iontophoresis device was filled with 200 μΐ of the OVA-liposome complex solution of Example 1. Next, the iontophoresis device was used. Mounted on the back skin of SD rats (10 weeks old, male), the rats were shaved with a shearing machine under Nembutal® anesthesia. Next, iontophoresis was carried out for 60 min at a constant current of 0.45 mA (0.15 mA/cm2). Administration of OVA-encapsulated liposomes The antigen-holding portion of the iontophoresis device (on the anode side) was filled with 200 μM of the OVA-encapsulated liposome solution of Example 1. Next, the iontophoresis device was mounted on the back skin of SD rats (10-week old, male) which had been shaved by a shearing machine under Nembutal® anesthesia. Next, iontophoresis was performed at a constant current of 0.45 mA (0.15 mA/cm2) for 60 min. Preparation of skin tissue sections and CLSM observations The skin was excised 3 hours after electrification, and the resultant was frozen and implanted for optimal cutting. Temperature (OCT) compound. Self-frozen skin samples were prepared using a cryostat to measure sections of 20 μηι and observed with a reverse confocal laser scanning microscope (LSM510 Carl Zeiss). Therefore, as shown in Fig. 2A, in the case where the OVA-encapsulated liposome was administered from the anode side, almost no fluorescence was observed in the epidermis layer or the dermis layer except for the stratum corneum. On the other hand, in the case where the OVA-liposome complex was administered from the cathode side, as shown in Fig. 2B, fluorescence was observed in the epidermal layer and the dermis layer. Test Example 3 138820.doc -21 · 200948395 Preparation of OVA encapsulated liposomes and OVA_lipid raft complexes OVA encapsulates were prepared using the same procedure as in Example 1 and Reference Example 1 using a non-fluorescently labeled OVA. Liposomes and OVA-liposome complexes. Percutaneous Immunization by Iontophoresis The prepared OVA-encapsulated liposome or OVA-liposome complex was administered to the dorsal skin of SD rats by iontophoresis in the same procedure as in Test Example 2. One week after the first administration, OVA-encapsulated liposomes or OVA-liposome complexes were again administered by iontophoresis in the same procedure 1. Blood was sampled 1 week after the second administration and serum was collected by centrifugation. Evaluation of the amount of anti-OVA antibody by ELISA The yield of the anti-OVA antibody in the obtained serum was evaluated by the following procedure by ELISA. 0.01 mg/ml OVA dissolved in carbonate buffer (pH 9·5) was added to a 96-well microtiter plate (nunc immunoplate maxisorp corting) at 50 μM/well, followed by incubation at 4 ° C overnight. Subsequently, the plate was washed 3 times with detergent buffer (-) (1 L PBS + 2.1 g NaCl) by using ImmunowashTM (BIO-RADTM), and a blocker (1% casein) was added at 150 μM/well. +1% gelatin + 0.5% BSA carbonate buffer solution), followed by incubation at 37 ° C for 1 h. Thereafter, the culture tray was washed 3 times with a washing buffer (+) (washing buffer (-) + 0.05% nucleus-X) in the same manner as described above. Next, serum samples each gradually diluted 10 to 20,000 times were added to the wells. After incubation at 37 ° C for 1 h, the plates were washed 3 times with wash buffer (+) in the same manner as described above. 50 μM of HRP-labeled secondary antibody was added to each well. After incubation at 37 ° C for 1 h, the plates were washed 3 times with wash buffer (+) in the same manner as described above for 138820.doc -22- 200948395, and 50 μM TMB was added to each well. Causes color development. After incubation at room temperature for 10 min, 50 μM of 1 N sulfuric acid was added to each well to stop the color reaction. Absorbance was measured at 450 nm with a micro-disc reader (BIO-RADTM). The anti-OVA was evaluated by subtracting the absorbance value in the serum of the untreated rat from the measured absorbance value and using the serum sample dilution (absorbance of 0.08) equal to or greater than the detection limit as an index. The production of antibodies. The results are shown in Figure 3. In the case where OVA-encapsulated liposomes were administered with β from the anode side, anti-OVA antibodies in serum were not detected at all dilutions (ND: not detected). On the other hand, in the case where the OVA-liposome complex was administered from the cathode side, an anti-OVA antibody was detected even at about 300-fold dilution (average soil standard deviation), thereby confirming the induction of antibody production. Test Example 4 1. Preparation of OVA-liposome complex The OVA-liposome complex was prepared by the same procedure as in Example 1 using a non-fluorescent-labeled OVA. 2. Percutaneous immunity by iontophoresis and monophospholipid A (MPL). After applying 100 μM of commercially available MPL adjuvant on the skin of the back of the shaved rat, the same as Test Example 2. The procedure is administered by iontophoresis with an OVA-liposome complex. The same test was performed but the MPL was not coated as a control. After 1 week of the second iontophoresis, blood was sampled and serum was collected by centrifugation. Evaluation of the production of anti-OVA antibody by ELISA method 138820.doc -23- 200948395 Antibody production was measured and evaluated by the same procedure as Test Example 3 by ELISA. The results are shown in Figure 4. In the case where the antigen was partially coated with MPL immediately before iontophoresis, the antibody production (average value) was about 4 times that of the unused MPL. Further, in the case of coating with MpL, even if the serum was diluted 1,000-fold or more than U00-fold, an anti-〇va antibody was significantly detected. The above description of the illustrated embodiments, including those described in the summary, are not intended to be While the invention has been described with respect to the specific embodiments and examples, it is understood that various equivalent modifications can be made without departing from the spirit and scope of the disclosure. The teachings of the various embodiments provided herein can be applied to other delivery methods without necessarily being the exemplary iontophoretic intradermal delivery method generally disclosed above. The various embodiments described above can be combined to provide other Example. In the event of inconsistency with the specific teachings and definitions herein, all US patents, US patent application publications, US special injections, foreign patents, and phase patents listed in this specification and/or application data sheet are included. The application and non-patent publications are fully incorporated herein by reference. If desired, the embodiments of the present invention may be modified to provide other embodiments by the various systems, processes, and concepts. These and other changes can be made to the embodiments in light of the above detailed description. In general, "T, in the scope of the following patent application, the terms used should not be construed as limiting the scope of the patent application to the specific embodiment disclosed in the specification of 138820.doc-24-200948395, but should be explained The equivalents of the full scope of the invention are included in all possible embodiments and the scope of the claims. Therefore, the scope of application for patents is not limited by the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing an iontophoresis device for administering an OVA-liposome complex according to an illustrative embodiment in an in vivo test; Fig. 2A is an OVA-liposome complex administration Reverse Confocal Laser Scanning Microscopy of Post-rat Frozen Skin Samples; ® Figure 2B is a reverse confocal laser scanning microscopy of rat frozen skin samples after OVA-encapsulated liposomes; Figure 3 shows and has been cast ELISA results relating to the production of anti-OVA antibodies in rat serum of OVA-liposome complexes or OVA encapsulated liposomes; and Figure 4 shows administration of monophospholipid A (MPL) and OVA-liposome complexes together with ELISA results related to the yield of anti-OVA antibodies in rat serum. [Main component symbol description] 1 Iontophoresis device 2 Power supply unit 3 Electrode assembly holding the composite 4 Electrode combination of the counter electrode as the electrode combination of the holding composite 5 Skin 6 Conductive path 7 Conductive path 31 Cathode 138820.doc -25 - 200948395 32 41 42 Protein-liposome complex retaining part of anolyte solution holding part 138820.doc -26-